中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (32): 6064-6067.doi: 10.3969/j.issn.1673-8225.2010.32.039

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

胰腺导管上皮细胞转分化为胰岛样细胞

赵艳艳，余  勤，李志臻，秦贵军   

  1. 郑州大学第一附属医院内分泌科，河南省郑州市  450052
  • 出版日期:2010-08-06 发布日期:2010-08-06
  • 通讯作者: 秦贵军，郑州大学第一附属医院内分泌科，河南省郑州市 450052
  • 作者简介:赵艳艳☆，女，1979年生，河南省新密市人，汉族，郑州大学医学院在读博士，主治医师，讲师，主要从事糖尿病并发症防治的研究。 Anny7997@163.com
  • 基金资助:

    河南省医学科技创新人才项目工程(2003-39)。

Transdifferentiation of mouse pancreatic ductal epithelial cells into islet-like cells

Zhao Yan-yan, Yu Qin, Li Zhi-zhen, Qin Gui-jun   

  1. Department of Endocrinology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou  450052, Henan Province, China
  • Online:2010-08-06 Published:2010-08-06
  • Contact: Qin Gui-jun, Department of Endocrinology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • About author:Zhao Yan-yan☆, Studying for doctorate, Attending physician, Lecturer, Department of Endocrinology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou 450052, Henan Province, China 7997@163.com
  • Supported by:

    the Medical Science and Technique Innovation Talent Project of Henan Province, No.2003-39*

PDF

507

被引次数

3

摘要：

背景: 胰岛移植是治疗1型糖尿病包括部分2型糖尿病的有效方法。然而，供体来源的匮乏及移植免疫排斥反应极大地阻碍了该方法的推广和应用。最有希望的来源是从干细胞诱导分化出大量可供移植的胰岛细胞，但目前胰腺干细胞转分化方面的研究尚处于起步阶段。
目的：寻找胰岛移植治疗糖尿病合适的细胞来源。
方法：分离培养昆明小鼠胰腺导管上皮细胞，以添加有角化细胞生长因子、肝细胞生长因子和烟酰胺的DMEM/F12培养基培养，不同时间取样本于光镜和电镜下观察，检测第1，16天时CK-19、PDX-1免疫化学染色变化并以半定量RT-PCR检测第1，16天时胰岛素和胰高血糖素基因表达情况，21 d时行双硫腙染色实验及葡萄糖刺激的胰岛素释放实验以检验胰岛样细胞的生理功能。
结果与结论：分离第1天，大部分细胞CK-19染色阳性，偶可见PDX-1阳性细胞，16 d后，CK-19阳性细胞快速增殖形成细胞团，大部分细胞PDX-1染色阳性；RT-PCR显示培养细胞胰岛素和胰高血糖素表达明显增强，分别增加了5.4倍和6.1倍(P < 0.01)；21 d时胰岛样细胞团更加成熟，双硫腙着色阳性，且对高糖(15 mmol/L)刺激的胰岛素释放较低糖(5.6 mmol/L)时增加了1.6倍(P < 0.05)。提示小鼠胰腺导管上皮细胞在体外培养条件下可增殖，并具有干细胞潜能，可转分化胰岛素分泌细胞。

关键词: 胰腺, 干细胞, 胰岛, 转分化, 小鼠

Abstract:

BACKGROUND: Islet transplantation is an effective method for the treatment of type 1 diabetes mellitus and parts of type 2 diabetes mellitus. However, its application is hindered by insufficient sources and immunologic rejection. Though transdifferentiation of pancreatic stem cells is at the starting step, it is thought to be the hopeful source for islet cell transplantation.
OBJECTIVE: To look for a suitable cells-transplantation source for the treatment of diabetes mellitus.
METHODS: The pancreatic ductal epithelial cells were separated from Kunming mice and cultured in DMEM/F12 medium supplemented with keratinocyte growth factor, hepatocyte growth factor and nicotinamide, etc. Samples were taken at different time points for light microscopy and electron microscope. The changes of CK-19 and PDX-1 were detected by immunocytochemistry at 1 and 16 days. The expressions of insulin and glucagon gene were detected by RT-PCR at 1 and 16 days. The physiologic function of these islet-like clusters was determined by dithizone staining and glucose stimulation at 21 days. 
RESULTS AND CONCLUSION: A large number of epitheliod cells were CK-19 immunoreactive positive and few of them were PDX-1 positive at 1 day after isolation, then CK-19 positive cells proliferated quickly and formed substantial plaques of epithelial cells in cobblestone pattern. At 16 days later, these cells begin to form islet-like clusters gradually, while most of them were PDX-1 immunoreactive positive. The analysis of mRNA by RT-PCR showed very low levels of insulin and glucagon mRNA in the starting materials but increase was found as the process of transdifferentiation. At 21 day differentiated islet-like clusters were stained red by dithizone. In those samples exposed to a stimulatory 15 mmol/L glucose, there was a 1.6-fold increase in insulin compared with to 5.6 mmol/L glucose (P < 0.05). Pancreatic ductal cells of adult Kunming mice could proliferate quickly and have the potency of transdifferentiation into islet-like clusters when cultured in vitro under appropriate conditions.

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